Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-monooxygenase.
7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000187:drug effects; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D011817:Rabbits; D013053:Spectrophotometry; D013253:Steroid 12-alpha-Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000302:isolation & purification*; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C035248:7 alpha-hydroxy-4-cholesten-3-one-12 alpha monooxygenase; D013253:Steroid 12-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital